In poultry nutrition, most attention is given to protein products, due to the importance of protein as a major constituent of the biologically active compounds in the body. It also assists in the synthesis of body tissue, for that renovation and growth of the body. Furthermore, protein exists in form of enzymes and hormones which play important roles in the physiology of any living organism. Broilers have high dietary protein requirements, so identification of the optimum protein concentration in broiler diets, for either maximizing broiler performance or profit, requires more knowledge about birds' requirements for protein and amino acids and their effects on the birds' growth performance and development. It also requires knowledge about the protein sources available that can be used in poultry diets. The broad aim of this review is to highlight the importance of some of the available high-quality specialized protein products of both animal and plant origins which can be explored for feeding broiler chickens. Minimization of the concentration of anti-nutritional factors (ANFs) and supplementation with immunologically active compounds are the main focus of gut health-promoting broiler diets. These diet characteristics are influenced by feed ingredient composition and feed processing. The general hypothesis is that these protein products are highly digestible and devoid of or contain less ANFs. Feeding these products to broiler chicks, especially at an earlier age, can assist early gut development and digestive physiology, and improve broiler growth performance and immunity.

This study was conducted to evaluate growth performance and ileal microflora in response to sugar cane bagasse (lignocellulose) and corn particle size. Three hundred and thirty six Ross 308 male broilers were assigned in a 2 × 2 factorial arrangement of treatments with 2 particle sizes (coarse and fine) and 2 levels of a lignocellulose (0%, 2%). Each treatment had 6 replicate pens of 14 birds from 0-24 d. Feed conversion ratio (FCR), weight gain and feed intake were measured from 0-10 and 10-24 d.

Wet litter is a recognized issue in commercial poultry production and litter quality has come under great scrutiny with the introduction of farming scheme standards by animal welfare organizations in Australia (RSPCA, 2013). According to RSPCA (2013) meat chicken standards, "litter must be maintained in a dry and friable condition." Litter conditions may affect odour emissions and thus litter properties were studied to correlate with specific odorants.

Odour emissions have been identified as a potential threat for the sustainable development of the broiler industry. Few of the methods examined to reduce odour emissions from poultry operations have proved effective. The methods including biofilters, litter treatments, neutralising agents, air scrubbers, ozone treatment are either impractical to apply or too costly to use in commercial farms. If diets could be formulated to more closely meet nutrient requirements, there would be reduced excretion of undigested components and lower level of substrates available for microbes to metabolize them to odorous compounds. Diet affects water intake, water to feed intake ratio, litter moisture, litter pH and litter water activity all of which may have an impact on the emission of odorants from the litter. This thesis details the role of diet composition, necrotic enteritis and litter condition on odour emission from broiler production. This thesis includes a comprehensive review on key odorants from broiler production, their origin, analytical techniques for odour measurements and nutritional factors affecting odour emissions. The five chapters that follow investigate the role of phytase enzyme (chapter 2), protein sources (chapter 3), protein levels (chapter 4), probiotic and saponin (chapter 4), litter condition (chapter 4 and 5), necrotic enteritis and high sodium diet (chapter 5) and the effect of Clostridium perfringens culture (chapter 6) on odour emissions. Nutritional strategies such as increasing meat meal in the diet, decreasing dietary protein levels and the addition of a Bacillus subtilis based probiotic and saponin blend may lower odour emission from broiler production. The results also indicate that odour is related to litter condition and if litter moisture content and water activity can be reduced and necrotic enteritis prevented, odour problems can be alleviated in broiler sheds.

The effectiveness of the addition of oat hulls (OH) as an insoluble fiber for improving nutrient digestibility and performance of birds fed diets containing full-fat canola seed (CS) was studied. A 2 × 2 × 2 factorial arrangement of treatments was used to assess the main effects of canola source (CS vs canola meal plus oil as control), OH (0 or 3%), pellet temperature (75 and 90°C) and their interactions. A total of 576 male day-old Ross 308 chickens were assigned to 8 experimental treatments, each replicated 6 times (12 birds per replicate). All birds were fed a same commercial starter diet for the first 10 d of age. Canola meal and canola oil in the control diets were replaced with CS at 11.6% and 13.5% in the grower (d 10 to 24) and finisher (d 24 to 35) diets, respectively. An interaction was observed between canola source and OH led to improved body weight gain (P < 0.01) and FCR (P < 0.05) in birds fed the combination of CS and OH in grower phase. Pelleting temperature at 75 vs 90°C did not affect performance of broilers. Birds fed diets containing OH had heavier gizzards at 24 and 35 d of age. Inclusion of CS in the diets depressed fat digestibility at d 24 (P< 0.001) and AME of the grower diets. At d 35, there was a significant interaction (P< 0.05) between CS and pellet temperature where birds fed CS diets pelleted at 75°C had higher fat digestibility than birds fed CS pelleted at 90°C. Regardless of canola source or pellet temperature, OH increased fat utilization at d 35 (P< 0.001) but had no effect on AME of the grower diets. In conclusion, CS can replace supplemental oil in broiler diets when an adequate source of insoluble fiber is included in the diet, which may help to maintain feed intake of broilers fed CS in steam-pelleted diets.

Necrotic enteritis (NE) is a major concern for the poultry industry worldwide as it causes reduced production and increased mortality, resulting in an estimated annual economic loss of US$6 billion (Wade and Keyburn, 2015). It is widely accepted that feed composition strongly influences the gut environment, consequently affecting the prevalence and severity of necrotic enteritis (Branton et al., 1997). Cereal grains such as wheat, barley and rye contain high levels of non-starch polysaccharides (NSPs) that have a direct negative impact on nutrient utilization. Insoluble NSPs can act as a physical barrier to enzymes. Soluble NSPs increase digesta viscosity, which may affect intestinal barrier function. Compromised barrier function results in translocation of luminal pathogens and a non-specific inflammatory response (Tellez et al., 2014).

The impact of two different strains of Clostridium perfringens (Cp) (NE-18 and NE-36) on mRNA expression of genes encoding proteins involved in cell apoptosis, intestinal tight junction and production of immunoglobulins and mucin in broiler chickens was investigated. A total of 468 male Ross 308 broilers was assigned to a 2 x 3 factorial arrangement of treatments. Factors were antibiotics (+/-), necrotic enteritis (NE) and challenge (-/ NE-18/ NE-36). A significant interaction between antibiotic treatment and challenge was observed on the expression of gene CASP3 (P < 0.05); NE-18 upregulated CASP3 only in the presence of antibiotics, while NE-36 upregulated this gene regardless of antibiotic supplementation. Interestingly, both NE strains upregulated CASP8 compared to the control treatment, but NE-36 showed higher CASP8 expression than observed with the NE-18 treatment (P < 0.001). It was shown that challenge significantly downregulated OCLD (P < 0.001), ZO-1 (P < 0.05) and MUC2 (P < 0.001). CLDN1 was significantly upregulated (P < 0.001) by both the NE-36 and NE-18 challenge, but NE-36 showing comparatively greater effects compared to NE-18. A significant antibiotic × challenge interaction on MUC5ac was observed (P < 0.05), where both NE- 18 and NE-36 upregulated MUS5ac when antibiotics was applied, but only NE-36 showed such upregulation in the absence of antibiotics. IgG and IgM were significantly downregulated (P < 0.001) by both strains of Cp. These results indicate that NE challenge alters the expression of genes investigated and different strains of Cp have varying impacts, which has a direct impact on severity of the disease.

Necrotic enteritis (NE), mainly caused by the gram-positive anaerobic bacterium Clostridium perfringens (Cp), is an enteric disease with great economic impact for the broiler industry. The prohibition of in-feed antibiotics has resulted in an increase in NE incidences globally. The severity of the disease in broilers depends on many factors. However, the type of Cp strains used plays a critical role. This study examined the impact of two Cp strains, i.e. NE18 and NE36, on performance, lesion score and gut permeability of broilers. Ross 308 broilers (n = 468) were assigned to a 2 × 3 factorial design (Feed: With/Without antibiotic) × (Challenge: No / NE18/ NE36). Oral administrations of Eimeria species (d9) and two stains of Cp (d14) were used to induce NE in the challenged groups. On d16 two birds from each pen were inoculated with FITC-d and serum samples were subsequently obtained for leaky gut evaluation. FITC-d amount was measured using a microplate reader (Synergy HT, Multi-mode microplate reader, BioTek Instruments, Inc., VT, USA). Broiler performance (Feed Intake, FI; weight gain, WG; and feed conversion ratio, FCR) was analysed for d0-24 period. Intestinal lesion scoring and gut permeability were measured at d16. Weight gain in the challenged group was significantly lower (P < 0.001) compared to the unchallenged birds at d24. Challenge also reduced the FI significantly (P < 0.001). FCR was significantly higher in the groups challenged with NE18 (P < 0.001) and NE36 (P < 0.001) compared to nonchallenged birds. Antibiotic diets significantly decreased FCR (P < 0.05) and increased WG (P < 0.05) in all groups. Birds challenged with NE36 showed higher FCR (P < 0.001) compared to the birds challenged with NE18 indicating a more severe impact of the NE36 than NE18. Ileal lesion scores and gut permeability were not significantly different between the two strains, but numerically higher number of lesions and gut permeability were observed in birds challenged with NE36. Supplementation with antibiotics reduced (P < 0.05) gut permeability in the birds of all groups. The findings of this study indicate that the severity of necrotic enteritis disease in challenge model can be modulated by the use of different Cp strains.

A 3-factor, 3-level Box-Behnken design was used to investigate the interaction effect of dietary digestible lysine (dLys, 9.5, 10.5, 11.5 g/kg), apparent metabolizable energy (AMEn, 12.77, 13.19, 13.61 MJ/kg) and available P (avP, 3.0, 4.0, 5.0 g/kg) levels on performance and amino acid (AA) digestibility of Ross 308 male broilers (n = 1,050) from d 14 to 34. The design consisted of 15 treatments each replicated 5 times with 12 birds per replicate. On d 34, 3 birds were sampled from each pen to collect ileal digesta (pooled per pen) to analyze AA. Response surface was fitted by first-, second-, or third-degree polynomial regressions in JMP statistical software v. 12.0.1. Feed intake (FI), weight gain (WG) and feed conversion ratio (FCR) were affected by dLys (linear and quadratic, P < 0.01), AMEn (linear, P < 0.01) and AMEn × avP (P< 0.01). Increased dLys increased FI but increased AMEn decreased FI in the birds fed the low-avP diet. However, when the avP level in the diet was increased, FI decreased to 13 MJ/kg AMEn and remained constant thereafter. Increased dLys increased WG whereas an increase in AMEn decreased WG in the birds fed the low-avP diet but had no effect on WG in those fed the high-avP diet. Increased dLys decreased FCR whereas increased AMEn decreased FCR in the birds fed the low-avP diet but had no effect on FCR in those fed the high-avP diet. Increased dLys increased breast yield percentage (linear, P < 0.01 and quadratic, P< 0.05) whereas increased AMEn decreased breast yield percentage (linear, P< 0.01). Dietary levels of dLys or avP had positive, linear effects on apparent ileal digestibility (AID) of methionine (P< 0.01) and threonine (P< 0.01) but had no effect on other AA (P> 0.05). These results indicate that increasing dLys levels above current industry standard would improve broiler performance irrespective of AMEn or avP levels of the diet.

Background: Count data generated by next-generation sequencing assays do not measure absolute transcript abundances. Instead, the data are constrained to an arbitrary "library size" by the sequencing depth of the assay, and typically must be normalized prior to statistical analysis. The constrained nature of these data means one could alternatively use a log-ratio transformation in lieu of normalization, as often done when testing for differential abundance (DA) of operational taxonomic units (OTUs) in 16S rRNA data. Therefore, we benchmark how well the ALDEx2 package, a transformation-based DA tool, detects differential expression in high-throughput RNA-sequencing data (RNA-Seq), compared to conventional RNA-Seq methods such as edgeR and DESeq2.
Results: To evaluate the performance of log-ratio transformation-based tools, we apply the ALDEx2 package to two simulated, and two real, RNA-Seq data sets. One of the latter was previously used to benchmark dozens of conventional RNA-Seq differential expression methods, enabling us to directly compare transformation-based approaches. We show that ALDEx2, widely used in meta-genomics research, identifies differentially expressed genes (and transcripts) from RNA-Seq data with high precision and, given sufficient sample sizes, high recall too (regardless of the alignment and quantification procedure used). Although we show that the choice in log-ratio transformation can affect performance, ALDEx2 has high precision (i.e., few false positives) across all transformations. Finally, we present a novel, iterative log-ratio transformation (now implemented in ALDEx2) that further improves performance in simulations.
Conclusions: Our results suggest that log-ratio transformation-based methods can work to measure differential expression from RNA-Seq data, provided that certain assumptions are met. Moreover, these methods have very high precision (i.e., few false positives) in simulations and perform well on real data too. With previously demonstrated applicability to 16S rRNA data, ALDEx2 can thus serve as a single tool for data from multiple sequencing modalities.

Motivation: Although seldom acknowledged explicitly, count data generated by sequencing platforms exist as compositions for which the abundance of each component (e.g. gene or transcript) is only coherently interpretable relative to other components within that sample. This property arises from the assay technology itself, whereby the number of counts recorded for each sample is constrained by an arbitrary total sum (i.e. library size). Consequently, sequencing data, as compositional data, exist in a non-Euclidean space that, without normalization or transformation, renders invalid many conventional analyses, including distance measures, correlation coefficients and multivariate statistical models.

Results: The purpose of this review is to summarize the principles of compositional data analysis (CoDA), provide evidence for why sequencing data are compositional, discuss compositionally valid methods available for analyzing sequencing data, and highlight future directions with regard to this field of study.

Gastrointestinal nematodes such as Ascaridia galli (A. galli) are prevalent in laying hens kept in production systems with free access to outdoor areas. A pilot study was conducted to establish an infection model with A. galli which then was used in two subsequent experiments to evaluate the impacts of different levels of A. galli infection on performance, egg quality and immune status of laying hens. In an artificial infection study, laying hens were inoculated with low (250), medium (1000), and high (2500) levels of embryonated A. galli eggs/hen. In a natural infection study, hens were ranged on the areas contaminated with A. galli during the artificial infection study. In both studies, uninfected hens served as control groups.

Infecting hens orally with 500 or 1000 embryonated A. galli eggs in multiple applications was found to be a more reliable method compared to infecting hens using a single dose. The artificial infection study showed that A. galli infection with different dose rates had no effect on feed intake, body weight, and FCR until 40 weeks of age. There was no difference in egg production at 25, 35 and 40 weeks of age. Serum and yolk antibodies against A. galli were higher at 20 weeks post infection.

Naturally infected hens had a higher infection intensity exhibited by higher intestinal worm counts and excreta egg counts compared to the artificially infected hens. However, this higher infection intensity did not negatively affect egg production, egg mass, feed intake, FCR and egg quality during the experiment. The liver lipid content of the hens with higher A. galli burden were compared to the uninfected hens and the results showed that the infected hens had consistently lower liver lipid reserves compared to the uninfected hens.

Across the two trials the sensitivity of the ELISA assay was 100% and 96% for serum and egg yolk samples respectively, whereas pooled excreta egg counts had sensitivity of 93%. Measurements of serum and yolk antibody levels can be helpful for the detection of current or prior A. galli infection. The practical and non-invasive method of yolk sample analysis is likely to be as informative as using serum samples to detect A. galli infection. Results of these experiments indicate that A. galli causes no production loss during the first three months of the free range laying hen's production cycle. However, production losses that might occur in the later stages of laying cycle still needs to be investigated.

Low protein (LP) diets have clear benefits for the poultry industry with regards to health, welfare and production. Dean et al., (2006) found that supplementing LP diets with glycine (Gly) and essential amino acids (AAs) can maintain performance similar to that seen on a standard protein (SP) diet. Further research into this concept proposes that supplementation of Gly precursors such as threonine (Thr) and serine (Ser) can overcome Gly deficiency in LP diets. Enzymes enabling these conversions include threonine dehydrogenase (TDH) and glycine C-acetyltransferase (GLYA) which convert Thr to Gly, and serine hydroxymethyltransferase (SHMT) which is responsible for the interconversion of Ser and Gly.

This study examined the effects of sources and levels of selenium (Se) on performance, carcass parts yield, meat quality and tissue Se concentration in broilers. A total of 960 one-day-old male broilers were divided into 8 treatments in a 4 2 factorial arrangement. Chicks were penned in groups of 20 with 6 pens per group. Selenium sources were sodium selenite (SS), Se enriched yeast (SY), DLselenomethionine (SM) and nano-selenium (NS) and dietary supplemental Se levels were 0.1 and 0.3 mg/kg diet. The average daily gain (ADG), average daily feed intake (ADFI), feed:gain ratio, mortality, and carcass parts yield were not affected by dietary treatments. The level of 0.3 mg/kg Se decreased lightness and increased yellowness of the breast and thighs (P < 0.001). Nano-selenium improved yellowness, redness and meat quality (P < 0.05). The interactive effects of sources and the levels of Se affected Se retention (P < 0.001). Inorganic Se showed poor retention compared to other sources of Se" and NS showed equal retention with the organic sources. With consideration to meat quality responses, NS had a more significant positive effect compared to SS as an inorganic source of Se. Overall, NS and organic sources of Se resulted in better meat quality compared with the inorganic source. Moreover, the highest Se retention percentage was achieved by supplementation of NS followed by organic sources at 0.1 mg/kg compared to SS.

Autism spectrum disorder (ASD) is a markedly heterogeneous condition with a varied phenotypic presentation. Its high concordance among siblings, as well as its clear association with specific genetic disorders, both point to a strong genetic etiology. However, the molecular basis of ASD is still poorly understood, although recent studies point to the existence of sex-specific ASD pathophysiologies and biomarkers. Despite this, little is known about how exactly sex influences the gene expression signatures of ASD probands. In an effort to identify sex-dependent biomarkers and characterize their function, we present an analysis of a single paired-end postmortem brain RNA-Seq data set and a meta-analysis of six blood-based microarray data sets. Here, we identify several genes with sex-dependent dysregulation, and many more with sex-independent dysregulation. Moreover, through pathway analysis, we find that these sex-independent biomarkers have substantially different biological roles than the sex-dependent biomarkers, and that some of these pathways are ubiquitously dysregulated in both postmortem brain and blood. We conclude by synthesizing the discovered biomarker profiles with the extant literature, by highlighting the advantage of studying sex-specific dysregulation directly, and by making a call for new transcriptomic data that comprise large female cohorts.

Global interest has emerged for the implementation of reduced-protein diets for meat chickens. The necessity of their development stems from environmental impacts and health and welfare concerns surrounding current meat-chicken production. Reduced crude-protein diets are possible with the increasing affordability of supplemental crystalline amino acids. Supplementing broiler feed with methionine, lysine and threonine is common practice in industry and has enabled a reduction of dietary crude protein to the levels currently used. However, further reduction of dietary protein often results in poor performance. Several nutritional options have been investigated with a focus on crystalline essential and non-essential amino acids such as glycine. However, reducing the crude protein of meat-chicken diets does change the ingredient and nutrient profile aside from the amino acid composition. Alterations in non-protein nitrogen concentrations, dietary electrolyte balance, minerals, fibre and carbohydrates, methyl-donors and polyphenols must be considered in formulations to ensure successful implementation of reduced-protein diets. The ability to maintain performance with reduced-protein diets may benefit sustainability and longevity of the meat-chicken industry.

Bone development is an important welfare and production concern in the broiler industry. It is affected by a number of factors including genetics and nutrition (González-Cerón, Rekaya, & Aggrey, 2015). Improved genetics resulting in higher growth and body weight places additional demands on nutrition to maintain skeletal structure. Calcium is one of the most limiting minerals in skeletal development. The presence of phytic acid in many ingredients is a major issue as calcium is easily chelated and forms phytate mineral complexes that reduce its availability. Furthermore, high levels of calcium reduce the ability of exogenous phytase to hydrolyse phytate.

Published literature indicates that L-arginine (L-Arg), guanidinoacetic acid (GAA) and L-citrulline (L-Cit) can all provide arginine activity in broiler diets (Su and Austic, 1999; Dilger et al., 2013; DeGroot, 2018) and all are commercially available. GAA has been reported to have 77% arginine equivalence for feed conversion (Ringel et al., 2013). This study was conducted to determine the effect of arginine deficiency on performance in reduced protein diets and the efficacy of adding it back as either L-Arg, GAA or L-Cit. Day old male Ross 308 birds (n = 768) were assigned to 8 dietary treatments using a completely randomized design: normal protein (NP, CP levels of 24.9, 23.7 and 21.4% for starter, grower and finisher, respectively), reduced protein deficient in Arg (RP, CP levels 5% lower than those of NP diets for all feeding phases) and RP with 2 levels of L-Arg (238 and 476 g/kg), GAA (309 and 618 g/kg) or L-Cit (238 and 476 g/kg).

Necrotic enteritis (NE) is an enteric disease of poultry caused by Clostridium perfringens (C. perfringens). The incidence of NE has increased in several countries as a result of restrictions on the use of in- feed antibiotics. This disease may be triggered by a combination of coccidiosis and the presence of undigested nutrients in the hindgut providing nutrients and high pH that favour the proliferation of patho-gens. Meat and bone meal (MBM) inclusion rates above 4% and higher dietary calcium (Ca) inclusion rates above 1.0% may potentially favour the overgrowth of enteric pathogens, including C. perfringens. High levels of elastin, collagen and keratin from MBM are refractory to gastric digestion and may act as nutrient substrates for C. perfringens. Such proteins are metabolised by C. perfringens by putrefactive fermenta-tion, producing trimethylamine and ammonia, that affect gut health and increase the pH of digesta. Digesta pH may become elevated by feeding a diet high in Ca, as this nutrient has a high acid-binding capacity. Calcium interacts with phytic acid, forming mineral-phytate complexes that decrease the activity of exogenous dietary phytase. This reduces digestion and increases the influx of nutrients into the hindgut. Similarly, smaller particle sizes (dgw <75 μm) and high solu-bility of some Ca sources could increase digesta pH and chelation of Ca by phytic acid and decrease nutrient digestibility. Whether the practice of overfeeding Ca in order to ensure this nutrient is not limiting for bone development may be putting the bird at risk of a NE outbreak is not known. This review discusses the potential for dietary MBM to exacerbate NE in broiler chickens. The impact of Ca level and particle size to affect gut pH and phytase efficacy are discussed, with inferences to alter the onset of NE.